A disintegrin and metalloproteases (ADAMs) belong the metzincin family of metalloproteases that also includes astacins and matrix metalloproteases (MMPs). They are all zinc-dependent metalloproteases. [Reiss, et al. Semin Cell Dev Biol (2009) 20, 126-137]. To date 23 human ADAMs have been identified. These are type I transmembrane multidomain proteins that contain a disintegrin and a metalloprotease domain that mediate cell adhesion via their disintegrin domain. Members of this family play important roles in many biological processes including cell-surface proteolysis, and cell-cell or cell-matrix interactions by virtue of their metalloprotease domain. They are major ectodomain sheddases that cleave a variety of cell surface proteins including cytokines, growth factors, receptors, as well as components of the extracellular matrix releasing soluble proteins. [White, Curr Opin Cell Biol (2003) 15, 598-606] This proteolysis can have multiple functional roles including the release of active peptides from proproteins for autocrine or paracrine signaling (e.g. TNFβ or EGFR ligands), soluble factors that act as antagonists to downregulate signaling, or intracellular proteins that act as transcription factors or indirectly modulate signaling pathways. Also, shedding has emerged as an important mechanism to decrease selective proteins from the cell surface and to inactivate receptors that could potentially act as decoys that sequester soluble ligands. [Reiss, et al. Semin Cell Dev Biol (2009) 20, 126-137; White, Curr Opin Cell Biol (2003) 15, 598-606]
ADAMs have been implicated in health and disease. They play critical role during fertilization, and are also important for cardiovascular and central nervous system development. They have been associated with autoimmune, inflammatory diseases and cancer progression when dysregulated. [Reiss, et al. Semin Cell Dev Biol (2009) 20, 126-137]
ADAM12, also called metrin alpha, is a member of the ADAM family of proteases. [Gilpin, et al. J Biol Chem (1998) 273, 157-166] The ADAM12 gene encodes two alternatively spliced transcripts; a long membrane-bound form that has the same structure as a typical ADAM protein (ADAM12-L) and a shorter secreted form that lacks the transmembrane and the cytoplasmic domains (ADAM12-S). [Gilpin, et al. J Biol Chem (1998) 273, 157-166] ADAM12-L consists of an amino-terminal secretion signal, prodomain, metalloprotease, disintegrin-like, cysteine-rich, epidermal-growth factor-like, transmembrane and cytoplasmic domains. Functionally, the signal peptide directs ADAM12 to the secretory pathway; the prodomain maintains the protease region in a latent form through cysteine switch mechanism. [Gilpin, et al. J Biol Chem (1998) 273, 157-166] The cytoplasmic domain contains several motifs involved in protein-protein interactions for intracellular signaling like SH3-containing domains. Deletion of the C-terminal amino acids accelerates the export of ADAM12 to the cell surface suggesting the presence of a retention signal in the cytoplasmic domain that functions as a limiting factor for the export of the protein from the ER. [Cao, et al. J Biol Chem (2002) 277, 26403-26411] ADAM12 is only active as a protease when the prodomain is cleaved. [Loechel, et al. J Biol Chem (1998) 273, 16993-16997] In fact, ADAM12 that lacks the prodomain is the predominant form at the cell surface. [Cao, et al. J Biol Chem (2002) 277, 26403-26411] Mutating the α-helical region in the prodomain (proline for leu73) results in retention of ADAM12 in the ER and lack of its proteolytic processing. [Cao, et al. J Biol Chem (2002) 277, 26403-26411] ADAM12 is synthesized in the ER and matures in the golgi compartment.
ADAM12 is expressed in mesenchymal cells and is lost in adult muscle. It reappears during tissue regeneration. ADAM12 knockout mice were viable and fertile with 30% embryonic lethality. Knockout and transgenic mice implicated ADAM12 in myogenesis [Yagami-Hiromasa, et al. Nature (1995) 377, 652-656] and adipocyte differentiation although the phenotype was mild with ADAM12 knockout mice is leaner than their control littermates with abnormalities in their brown adipose tissue with a reduction in the number of adipocytes. [Kawaguchi, et al. Am J Pathol (2002) 160, 1895-1903]
ADAM12 is implicated in a variety of diseases including muscular dystrophies, Alzheimer's (where two single nucleotide polymorphisms in the ADAM12 gene were identified and significantly associated with late-onset Alzheimer's disease, [Harold, et al. Am J Med Genet B Neuropsychiatr Genet (2007) 144B, 448-452] and processes characterized by excessive growth including pregnancy where it is highly expressed in human placenta and reach high concentrations in maternal serum from the first trimester, and is currently being evaluated as a prenatal marker for the detection of chromosomal abnormalities as ADAM12-S serum concentrations are significantly reduced in trisomy-18 [Spencer, et al. J Matern Fetal Neonatal Med (2007) 20, 645-650] and trisomy-21 pregnancies [Toning, et al. Reprod Biol Endocrinol 8, 129], cardiac hypertrophy [Wang, et al. Circulation (2009) 119, 2480-2489] but most importantly in cancer being upregulated in a variety of human tumors. [Kveiborg, et al. Int J Biochem Cell Biol 2008 40, 1685-1702] It is suggested to be involved in the development and progression of tumors possibly due to stimulating growth factor responses by a mechanism called “shedding” that releases active proteins, and also through its adhesion activity. Interestingly, ADAM12 was also linked to CNS inflammation by its upregulation in the spinal cords of mice with EAE, being expressed primarily by infiltrating T cells. [Toft-Hansen, et al. J Immunol 2004 173, 5209-5218] However, not much is known about the function of this gene in T cells and more specifically in Th17 cells especially that increased levels of IL-17 transcripts were associated with multiple sclerosis and the severity of EAE in mice.
It was shown that ADAM12 regulates TGFβ signaling through its interaction with TGFbRII where the two proteins localize to areas near the cell surface. [Atfi, A. et al. J Cell Biol (2007) 178, 201-208] ADAM12 increases the steady state level of TGFRII and decreases its ubiquitination and turnover, resulting in increased SMAD2 and SMAD3 phosphorylation and decreased SMAD7. ADAM12 does not maintain transcriptional activity after TGF-β removal indicating a positive feedback mechanism between TGF signaling and ADAM12. [Atfi, A. et al. J Cell Biol (2007) 178, 201-208] In support of this notion, it was shown that TGFβ signaling causes derepression of the ADAM12 gene in a SMAD2/3 dependent manner, through inducing the proteasomal degradation of SnoN, the repressor that negatively regulates ADAM12. [Solomon, et al. J Biol Chem 285, 21969-21977] Overexpression and silencing of SnoN alters the magnitude of ADAM12 induction by TGFβ. [Solomon, et al. J Biol Chem 285, 21969-21977] However, still not much is known regarding the regulation of ADAM12, possible through hormones, cytokines and growth factors.
TGFβ is a secreted protein that exists in three isoforms called TGFβ1, TGFβ2, and TGFβ3. It was also the original name for TGFβ1, which was the founding member of this family, and the predominant form expressed in the immune system. TGFβ is synthesized as a precursor made of a dimer of mature TGFβ, that is non-covalently linked to a dimer of latency associated peptide (LAP) in a complex called small latent complex (SLC). LAP in SLC binds one molecule of latent TGFβ binding protein (LTBP) to form a bigger complex called large latent complex (LLC). LLC is released from the cell and needs further processing to deliver active TGFβ. [Todorovic, et al. Int J Biochem Cell Biol (2005) 37, 38-41]
TGFβ resists proteolysis and extreme pH conditions, and so the most common laboratory way to activate TGFβ is by treating media or biological fluids with an acid that lowers the pH to 2.0 for a short period of time. [Taylor, J Leukoc Biol (2009) 85, 29-33] Alternatively, incubating culture media for 10 minutes at 80° C., or by freezing and thawing can activate TGFβ. The in vivo activation mechanisms are still speculative, but could involve proteases that cleave LTBP and frees LAP in LLC from the matrix. LAP now can be further cleaved by proteases or through conformational changes to release mature TGFβ. [Taylor, J Leukoc Biol (2009) 85, 29-33]
There are different groups of TGFβ Receptors. The first one binds LAP as part of LLC or SLC and includes integrins αvβ6 and αvβ8 [Wipff, et al. Eur J Cell Biol (2008) 87, 601-615], thrombospondin-1 and neuropilin [Glinka, et al. J Leukoc Biol 2008 84, 302-310], as well as tissue matrix proteins that bind LTBP. These surface receptors of LAP allow the cells to hold latent TGFβ on their surface for autocrine or paracrine signaling of active TGFβ. This is suggested as one mechanism by which regulatory T cells suppress the activity of other T cells. [Glinka, et al. J Leukoc Biol 2008 84, 302-310] Tregs express LAP on their membrane surface [Chen, et al. J Immunol (2008) 180, 7327-7337] and LAP+Tregs produce active TGFβ. Neuropilin is so far the identified binding protein for LAP on Tregs and could be the way by which Tregs activates TGFβ and delivers it to other T cells to suppress immune responses.
The second group of receptors binds active TGFβ. [Massague, Mol Cell (2008) 29, 149-150] This comprises TGFβRI, TGFβRII, and TGFβRIII. Type III acts as a sink, mopping up active TGFβ; however, when bound on the cell surface, it facilitates TGFβ binding to RII. Active TGFβ binds RII, which recruits and phosphorylates RI. An activation complex is formed and made of 2 pairs of RI and RII, with each pair binds one of the two chains of active TGFβ. [Massague, Mol Cell (2008) 29, 149-150] Phosphorylated RI then recruits and phosphorylates a receptor regulated SMAD (R-SMAD) like SMAD2 and SMAD3. R-SMAD then binds to SMAD4 and forms a heterodimeric complex that then enters the nucleus where it acts as a transcription factor for many genes. SMAD pathway is the canonical signaling pathway that TGFβ family members signal through to target genes.
TGFβ is a pleiotropic cytokine involved in various physiological and pathological processes such as carcinogenesis and embryogenesis. [Blobe, et al. N Engl J Med (2000) 342, 1350-1358] In the immune system, recent studies have defined TGFβ as a critical regulator of thymic T cell development, an important player in T cell homeostasis, peripheral tolerance to self-antigens to limit inflammatory diseases and in T cell differentiation particularly Th17 and Tregs. [Li, et al. Cell (2008) 134, 392-404]
The induction of Th17 and Tregs by TGFβ is mutually exclusive meaning that the conditions that favor Th17 inhibits Tregs and vice versa. During homeostasis, the development of induced Tregs is favored in the TGFβ-rich gut-associated lymphoid tissue (GALT) promoted by retinoic acid, a metabolite of dietary vitamin A produced by dendritic cells in the intestinal mucosa. When pathogenic bacteria activate DCs, the latter produce inflammatory cytokines, do not metabolize vitamin A to retinoic acid, and therefore TGFβ-induced differentiation of naïve T cells is diverted away from Tregs to Th17 cells. [Bettelli, et al. Nature 2006) 441, 235-238; Mangan, et al. Nature (2006) 441, 231-234]
The importance of TGFβ in T cell lineage development is supported by evidence that mice with αvβ8 (activator of TGFβ) knocked out on their dendritic cells suffer from autoimmunity and colitis. [Travis, et al. Nature (2007) 449, 361-365] There is a significant loss in the ability of these DCs to induce activation of Treg, and that was reversed by the addition of active TGFβ. [Travis, et al. Nature (2007) 449, 361-365] The fate of Th17 cells was not examined in this study, but one may speculate that αvβ8 mice have high levels of IL-17 producing cells, which potentially contribute to the immunopathology seen in these mice.
Gruel et al., BMC Research Notes 2009; 2:193 teach that ADAM12 induces a permanent response to TGFβ in some cells. Wewer et al., WO2005/111626 teach a method, an assay and a kit for providing an indication of abnormal cell function based upon change in serum ADAM12 concentration. ADAM12 was described as an overall general marker for abnormal cell function, and an important indicator of fetal chromosomal disease and placenta function, e.g. Downs's syndrome, trisomy 18, preeclampsia, and Turner syndrome in both first and second trimester.
Bosch et al., WO2006/014903 teach that ADAM12L is overexpressed on the surface of cancer cells compared to normal tissues and therefore is a therapeutic target for treating cancer. Modulators of ADAM12, highly expressed in cancerous tissue compared to normal tissue are indicated as useful for treating certain proliferative disorders such as cancer and psoriasis.
Suonpaa et al., WO2008/119882 teach epitopes of ADAM12 and to binding agents specific to those epitopes. Also, Suonpaa et al. teach methods of detecting ADAM12 in a biological sample as well as diagnostic and screening methods using these binding agents.